Theme C: Spatial Control of Intracellular Signaling Theme Leader: Bard well Other Project Faculty: Jeon, Komarova, Liu, Nie, Yi The proper growth, development and survival of an organism requires extensive and accurate communication between that organism's cells. Accordingly, cells react to a wide variety of stimuli, which deliver information about nutrients, harmful insults, the state of neighboring cells, etc. Many incoming stimuli are first recognized by a cell surface receptor, and then transmitted to various locations inside the cell by a cascade of signaling proteins. Such 'signal transduction pathways'are the connection between signal and cellular response. Accurate and efficient signal transduction is challenging because (i) there are multiple inputs which must be routed to the correct output, (ii) the inputs vary in time and space, (iii) noise is ubiquitous, and (iv) pathways may share similar or identical components. The central hypotheses of this theme are (1) that accurate and efficient signal transmission requires sophisticated regulation of the spatial dynamics of the signaling network;(2) that achieving specificity from signal to cellular response in a highly interconnected network requires the cooperative action of multiple insulating mechanism (some of which depend upon or exploit spatial aspects). To investigate these hypotheses we propose to examine the relationship between the spatial control of signaling and directional sensing (Project C.1), how different network architectures can enhance specificity (Project C.2), the performance objectives of the transcriptional response to a spatial signal (Project C.3) and the role of spatial localization in signal transmission and specificity (Project D.4) Utility of yeast mating pathway as a model system. We will focus our combined theoretical and experimental effort on the mating pheromone response pathway of the yeast S. cerevisiae, because it is one of the best understood signaling pathways in eukaryotes, and is thus suited for attempts at systematic understanding. Haploid yeast cells respond to the presence of peptide mating pheromone by undergoing a series of events resulting in fusion with a nearby mating partner [reviewed by Bardwell, 2005]. Pheromone binds to a seven-transmembrane, G-protein-coupled receptor (Ste2) resulting in the activation of a membrane-bound heterotrimeric G-protein (see Figure C1). Activated G(3 (Ste4) then binds to the Ste5 adapter/scaffold and to the Ste20 kinase. As a result, Ste20 phosphorylates and activates the Ste11 kinase, which has been towed to the membrane by Ste5. Ste11 is the topmost kinase in a prototypical mitogenactivated protein kinase (MAPK) cascade: Ste11 (MAP3K. or MEKK) phosphorylates Ste7 (MAP2K, or MEK), which in turn phosphorylates Fus3 and Kss1 (MAPKs). Signal transmission down the MAPK cascade is enhanced by the Ste5 scaffold protein, which binds to Ste11, Ste7 and Fus3, and is thought to tether these kinases in a close and productive configuration. Fus3 and Kss1 then phosphor/late several substrates, including the Dig1, Dig2 and Ste12 transcriptional regulators. As a result, Dig1/2 dissociate from Ste12, and Ste12 activates a battery of genes required for cell fusion. Additional components of this pathway include a Cdc42 G-protein cycle and associated regulators. MAPK cascades and specificity. MAPK cascades are found in all animals, plants and fungi, where they participate in the regulation of normal and pathological aspects of cell growth, division, differentiation, and death (Johnson and Lapadat, 2002). How are MAPK cascades used so ubiquitously and versatilely yet in a way that maintains specificity? How do different signals elicit distinct responses when they are transmitted by the same components? Yeast is and outstanding model to study such questions, because elements of the same MAPK cascade that regulate mating also regulate two other distinct responses - the filamentous invasive growth differentiation program, and the HOG stress response pathway (see Fig C2). Mating and filamentous invasive growth are both regulated by Ste2QPAK, ste11MEKK and Ste?MEK |n addition, Ste2QPAK and Ste11 MEKK are also activated during osmotic stress. As stated above, FusSMAPK and Kss1MApK are activated during mating. In addition, Kss1 is also activated during invasive growth (HoglMAPK is activated during the stress response). Despite this sharing of key components, the three pathways are well insulated from one another: exposure of cells to mating pheromone does not result in the hyperactivation of filamentation or stress response genes, for example. How this is achieved is an area of active investigation (Schwartz and Madhani, 2004;Bardwell, 2006), and is one of the major emphases of this Theme. Modeling signal transduction. A quantitative understanding of intracellular signal processing will substantially increase our understanding of biological systems and may catalyze radical changes in how diseases are understood and treated. Hence, there has recently been a growing effort to analyze the flow and processing of information within cells (reviewed by Endy and Brent, 2001;Ferrell, 2002; Neves and lyengar, 2002;Schuster et al., 2002; Kholodenko, 2003;Wiley et al., 2003;Sauro and Kholodenko, 2004;Orton et al., 2005). For the most part, theoretical treatments of kinase signaling have used ordinary differential equations, and have not explicitly considered spatial dynamics. These efforts have also not explicitly considered important aspects of signaling specificity. It is these elements that we focus on here.